Mesenchymal stem cells (MSC) are multipotent cells that can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells). This capability has been demonstrated for specific cells and tissues in living animals and for their counterparts grown in tissue culture.
MSC are a valuable resource for cell-based drug discovery and a potential therapeutic for a number of human diseases. The potential of MSC to treat disorders including graft-versus-host disease, multiple sclerosis, Crohn's disease, type 1 diabetes, bone fractures and osteoarthritis is currently being examined in clinical settings.
It is estimated that MSC comprise a mere 0.001 to 0.01% of total bone marrow mononuclear cells; therefore, this population requires extensive in vitro cell culture expansion to obtain sufficient numbers for basic biological or clinical applications. Current clinical applications use an approximate dose of 100 million cells.
Current developments in regenerative medicine and cellular therapy suggest that there will be an extraordinary increase in cell processing technology in the coming years. This will relate to adult and embryonic stem cells as well as somatic cells used for therapeutic purposes. In addition, the increased use of stem cells and induced pluripotent stem cells in drug screening applications will see the demand for cell processing technology increase greatly.
Isolation and culture of these MSC relies for the most part on the use of animal products, in particular foetal bovine serum (FBS).
Stem cells require specific growth conditions. Currently, bone marrow-derived MSC are isolated, cultured and differentiated in serum-containing medium.
Virtually all mammalian cells grown in laboratory culture require the presence of FBS in the growth medium, which provides hormones and growth factors necessary for cell attachment and proliferation. This includes human cells currently under investigation for therapeutic use, such as adult and embryonic stem cells and other somatic cells. FBS is harvested from the blood of foetal calves obtained in the meat processing industry and is generally added to growth media at a concentration of 10% by volume. Consequently, the supply of stem cells is dependent on the availability of suitable media to grow them in.
This technique has been successfully applied for several decades and FBS is the most commonly used growth medium supplement because of the complex mixture of proteins and high levels of growth-stimulatory factors present in foetal blood. However, there are problems associated with the use of FBS, some of which are listed below:                1. Risk of disease transmission to recipients of cellular therapy        2. Risk of disease transmission to processing technicians and medical personnel        3. Uncertain and seasonal availability of product        4. Need for cryostorage at −20° C.        5. Significant batch-to-batch variability.        6. Anticipated major supply-chain problems because of increased demand.        
A major concern associated with the use of FBS in cells produced for human therapy is the risk of transmission of infectious material to the recipient. In addition there is considerable risk associated with the handling of these materials by processing technicians and medical staff. This risk has become starkly serious in recent years because of the emergence of transmissible bovine spongiform encephalopathy (BSE), commonly known as mad-cow disease, and the associated risk to humans of acquiring variant Creutzfeldt-Jakob disease (vCJD). To date, BSE has been detected in cattle in Europe, North America and in many other parts of the world. Almost all FBS now approved for use in the growth of cells for human therapy comes from Australia and New Zealand, which are still BSE free.
The emergence of BSE in cattle herds throughout the world has also lead to a major supply problem, and this is predicted to have a serious impact on the availability of cellular therapies in the future. Currently this limited supply of serum is generally sufficient for laboratory and research use. However, some of these laboratory and research uses are directed to producing a commercial product that will be produced on a medium or large scale. Currently there are no applications for stem cell technologies that are approved for general use. Thus, it can be envisioned that when a stem cell technology is released for general use the supply of serum will simply not be adequate to meet this demand.
Indeed, it is anticipated that, when cellular therapy products are approved for just two major indications, the entire global supply of approved, BSE-free FBS will be consumed within the first year of manufacturing. Thus, as cellular therapies gain regulatory approval and are used there will be an extraordinary and unprecedented demand for FBS-free growth medium. Clearly, the supply of stem-cell growth medium will be a limiting factor in the application of stem-cell technologies.
In addition, FBS is a heterogeneous mixture, which leads to a lack of reproducibility and performance between serum batches. Serum contains many unidentified or non-quantified components and therefore is not “defined” and cannot be recreated synthetically. Consequently, the composition of serum varies from batch-to-batch, making standardization difficult for experimentation or other uses of cell culture. Also, MSCs are an adherent cell population and currently this capacity to adhere to tissue culture plastic relies on FBS for the most part.
Indeed, because many of the components of FBS affect cell attachment, proliferation and differentiation, controlling these parameters, or studying the specific requirements of cells with respect to these parameters, is precluded by the use of serum. Furthermore, some components of serum are inhibitory to the proliferation of specific cell types and to some degree may counteract its proliferative effect, resulting in sub-optimal growth.
The variability in growth characteristics caused by the heterogeneous nature of FBS leads to a lack of reproducibility between serum batches and presents problems for growth and manufacturing of cell-based products and therapies. This is particularly the case for products made under Good Manufacturing Protocols (GMP) which are presupposed to operate using consistent, and therefore reliable, starting materials.
Furthermore, from a therapeutic and regulatory standpoint, the presence of FBS is not ideal as this may lead to the transfer of animal pathogens. While serum is approved for medical use, it may contain viruses which may affect the outcome of experiments or provide a potential health hazard if the cultured cells are intended for implantation in humans. Furthermore, from a regulatory point of view, the use of animal-derived products in a clinical setting is undesirable not only because of the risk of transmission of infectious agents but also the potential to evoke an immune response.
Thus there is a need for a growth medium for the propagation and/or production of stem cells that does not need to be supplemented with serum. Any such new growth medium should preferably be relatively simple and easy to produce in large quantities.
Consequently, efforts have been made to eliminate FBS from culture media and develop a defined serum free medium capable of ex vivo expansion of MSC.
WO 2011/111787 discloses a cell preparation that contains mesenchymal stem cells which maintain immunosuppressive ability, produced by serum-free or low-serum culture. A method for producing a cell preparation containing mesenchymal stem cells is described that comprises: a proliferation step wherein mesenchymal stem cells are proliferated in a culture medium that contains FGF, PDGF, TGF-β, HGF, EGF, at least one phospholipid and at least one fatty acid; and a screening step wherein the mesenchymal stem cells after the proliferation step are screened for mesenchymal stem cells in which the immunosuppressive ability is maintained or improved.
WO 98/104681 discloses a serum-free defined cell culture medium comprising a supplement mixture, a component mixture, a vitamin mixture, an inorganic salt mixture and an amino acid mixture. This defined medium is disclosed as being useful for culturing fibroblasts, especially chondrocytes. Also disclosed is a method of enhancing the differentiation of chondrocytes and enhancing the synthesis of a cartilage specific matrix using tumor growth factor beta (TGF-β) and a method of enhancing the differentiation of chondrocytes using the combination of TGF-β and insulin-like growth factor (IGF).
Other media for MSC have been proposed. WO 2005/113751 describes expansion of MSC in serum-free media; the media are highly complex and some can contain, inter alia, bFGF and TGF-beta. US 2010/0279412 also describes serum-free media, which may contain a FGF, a TGF, an EGF, phosphatidic acid, phosphatidylcholine and vitamin C. Chase et al, Stem Cell Research & Therapy 2010, 1:8 describe a serum-free medium for human MSC, containing bFGF, TGF-beta1 and PDGF.
It is an object of the present invention to provide a medium and methods for the growth of stem cells that represents at least an alternative to the above, and preferably solves or at least ameliorates one or more problems identified in the prior art. An object of specific embodiments is to provide a medium for the growth of MSC that does not require supplementing with serum and thus is easily available and relatively inexpensive.